Method of treating cancer that overexpresses TopBP1

ABSTRACT

The present invention provides a method of treating cancer that overexpresses TopBP1 by administering to a patient suffering from the cancer with an effective amount of a small molecule inhibitor that binds the BRCT7/8 domain of TopBP1.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication Ser. No. 62/068,918, which was filed on Oct. 27, 2014.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to treating cancer that overexpressesTopBP1 by administering to a patient suffering from the cancer with aneffective amount of a small molecule inhibitor that binds the BRCT7/8domain of TopBP1.

2. Description of the Related Art

Despite the complexity of mutations in different cancers, recent effortsin cancer genome sequencing project have shown a handful of coresignaling pathways, such as receptor tyrosine kinases(RTK)/RAS/phosphatidylinositol 3-kinase (PI(3)K), p53 and retinoblastoma(Rb) protein, are deregulated in majority of solid tumors. For example,77% of breast cancers have genetic alterations in PI(3)K/Akt pathway,and 49% have alterations in p53 signaling (see reference in (TCGA2012)). Particularly in basal-like breast cancer (often triple-negativebreast cancer or TNBC), 84% show TP53 mutations, 35% show PTENmutation/loss and 20% show RB1 mutation/loss (see reference in (TCGA2012)). These deregulated signaling pathways often converge to somecommon modulators.

As a key regulator for cell growth, the Rb pathway is de-regulated inmost cancers, resulting in high E2F1 activities to drive cell cycleprogression. E2F1 also has a pro-apoptotic role through activatingtarget genes such as p73 (Irwin et al. 2000; Stiewe and Putzer 2000),Apaf-1, and caspases (Muller et al. 2001; Nahle et al. 2002) during DNAdamage (Lin et al. 2001). How to activate E2F1 pro-apoptotic activityinside cancer cells remains an elusive goal. Previously, we showed thata checkpoint activator protein, TopBP1 (topoisomerase IIβ-bindingprotein 1), plays a critical role in suppressing E2F1 pro-apoptoticactivity in response to PI(3)K/Akt signaling, which suggests TopBP1 as atherapeutic target to activate E2F1-dependent apoptosis in cancer (Liuet al. 2003; Liu et al. 2004; Liu et al. 2006; Liu et al. 2013).

TopBP1 utilizes its multiple BRCA1 carboxyl-terminal (BRCT) motifs asscaffolds to modulate many processes of DNA metabolism; such as DNAdamage checkpoint, replication, and transcription (Garcia et al. 2005).TopBP1 represses E2F1 transcriptional activities by recruiting Brg1/Brmchromatin remodeling complex (Liu et al. 2004). TopBP1 also binds theDNA-binding domain (DBD) of p53 to inhibit its transcriptional function(Liu et al. 2009). Regulation of E2F1 and p53 by TopBP1 is important tocontrol the pro-apoptotic activities of both transcription factorsduring normal S phase transition. While TopBP1 is involved in seeminglyseparate functions, our recent study showed that its functions inreplication checkpoint and transcriptional regulation are indeedcoordinated via an Akt-dependent conformational change of TopBP1 (Liu etal. 2013). Akt phosphorylates TopBP1 at Ser1159 and induces itsoligomerization through an intermolecular interaction between thephosphorylated Ser1159 residue (pS1159) and the 7^(th)-8^(th) BRCT(BRCT7/8) domain of two TopBP1 molecules (Liu et al. 2006; Liu et al.2013). Oligomerization of TopBP1 then induces its binding to E2F1, butat the same time prevents its recruitment to chromatin and ATR bindingand perturbs its checkpoint-activating functions (Liu et al. 2013).Thus, by regulating TopBP1 quaternary structure, Akt switches TopBP1function from checkpoint activation to transcriptional regulation. Thismechanism is responsible for inhibition of E2F1-dependent apoptosis (anoncogenic checkpoint) and inhibition of ATR function (replicationcheckpoint) in the tumors with high Akt activity. Therefore, selectiveblockade of TopBP1 oligomerization may provide a novel therapeuticstrategy in cancer cells which exhibit up-regulated PI3K/Akt signaling.

Many mutant p53 (mutp53) proteins do not only lose normal p53 function,but also gain new functions which contribute to cancer progression(“gain of function” activities (GOF)) (Freed-Pastor and Prives 2012;Muller and Vousden 2013). In addition to mutations, cancer cells canhave a different mechanism to inactivate p53: up-regulation of p53negative regulators, such as MDM2 (Manfredi 2010), MDMX (Marine et al.2006) and TopBP1 (Liu et al. 2009). Adding to the complexity of p53regulation is the presence of 12 p53 isoforms with differentialexpression (Khoury and Bourdon 2011), some of which havedominant-negative activities against p53 (Bourdon et al. 2005).

TopBP1 also mediates mutp53 GOF by facilitating its complex formationwith NF-Y and p63/p73 (Liu et al. 2011). Since TopBP1 is an E2F target(Liu et al. 2004), it is often up-regulated upon inactivation of the Rbpathway (Liu et al. 2009). Indeed, TopBP1 is frequently overexpressed inbreast cancer and its overexpression is associated with a shortersurvival (Liu et al. 2009; Liu et al. 2011). SNPs in TopBP1 that causehigher expression of TopBP1 mRNA and protein have also been associatedwith an increased risk in breast and endometrial cancers (Forma et al.2013a; Forma et al. 2013b). Thus, deregulation of the Rb pathway may befunctionally linked to mutp53, and be responsible for at least part ofmutp53 GOF via TopBP1. The accumulated TopBP1 in cancer cells theninhibits growth checkpoints through repressing E2F1 and p53 functions,and collaborates with mutp53 to further promote tumor progression.Therefore, TopBP1 might be a target among the nexus of these majoroncogenic pathways. Here we perform a molecular docking screening andidentify calcein as a compound to target the BRCT7/8 domain of TopBP1.We also use its cell-permeable derivative Calcein AM to provideproof-of-principle evidence for targeting TopBP1 as a cancer therapy.

SUMMARY OF THE INVENTION

We identify a lead compound targeting C-terminal BRCT domains of TopBP1,and provide evidence to support TopBP1 as a novel cancer therapeutictarget that functions in a convergent point of multiple oncogenicpathways: inactivation of Rb pathway leads to its overexpression, andactivation of RTK/RAS/PI(3)K induces its phosphorylation. Theaccumulation of phosphorylated TopBP1 in cancer cells may cause defectsin growth checkpoints through repressing E2F1 and p53 functions, andturning mutp53 into oncoproteins (FIG. 10b ). Using a small moleculeinhibitor capable of blocking the endogenous interactions between TopBP1and E2F1 as well as mutp53, we validate TopBP1 as a therapeutic targetin the tumors to activate E2F1 pro-apoptotic activity and to avert theoncogenic activity of mutp53. While the cytotoxic activity of Calcein AMagainst cancer cells was noticed before, we now identify its in vivotarget and elucidate its selective anti-tumor activity through a novelTopBP1-dependent mechanism of action. Finally, for the first time wedemonstrate Calcein AM antitumor activity in two breast cancer xenograftmodels. While drug development typically aims at upstreamreceptors/kinases, our study provides evidence for targeting theproteins in the nexus of multiple signaling pathways as new cancertherapeutics.

Different mutant p53 proteins may have different functions. TopBP1 canbind to several hot-spots mutants, including both DNA contact mutant(such as R273H) and conformational mutants (such as R175H and R249S)(Liu et al. 2011). Consistently, by targeting TopBP1, Calcein AMreleases p73 from mutp53(R273H) in MDA-MB468 cells and frommutp53(R249S) in BT549 cells. Although the effect of Calcein AM againsteach mutant p53 will need to be tested experimentally, the two examplespresented here suggest that Calcein AM might be used to treat cancercells harboring a broad spectrum of p53 mutations. In BT549 cells, wedemonstrated a role for E2F1 and p63/p73 for mediating CalceinAM-induced apoptosis (FIG. 7). TopBP1-BRCT7/8 also binds to othertranscription factors, such as Miz1 (Herold et al. 2002; Liu et al.2006). In fact, Calcein AM treatment also released Miz1 from TopBP1(FIG. 2f ), suggesting that Miz1 might also contribute to the toxiceffects of Calcein AM. The other factor worth considering is thedifference in genetic alterations causing Akt activation in cancer. PTENloss mainly occurs in basal-like breast cancer, whereas PIK3CA mutationsare more common in luminal or Her2E subtypes breast cancer (seereference in (TCGA 2012)). Both MDA-MB468 and BT549 cell lines testedhere have PTEN loss. Whether cancer cells with PIK3CA mutations responddifferently to Calcein AM will be tested in the future. According toNCI-60 cell lines database, a PIK3CA mutant (H1047R) cell line T47Dstill responds to Calcein AM (GI₅₀ 1.4 μM) (FIG. 6a ).

Calcein AM has been considered to be non-toxic to a wide spectrum ofcell types esp. in normal cells, thus is widely used for viability assayand in vivo physiological cell marking. The fact that the mice injectedwith Calcein AM did not manifest any apparent toxicity in our studyindicates that the dose (40 mg/kg) that is effective in shrinkingMDA-MB468 xenografts is quite tolerable for the mice. Consistently, thetoxicology study of Calcein AM performed by Dojindo Laboratories showsthat oral LD50 in rats is 14.5 g/kg; oral LD50 in mice is 17 g/kg;percutaneous LD50 in mice is 5 g/kg (according to Material Safety DataSheet,http://www.dojindo.com/store/p/162-Cellstain-Calcein-AM-Solution.html).On the other hand, calcein may bind ions such as Ca and Mg, thus thepotential effects of ion chelation need to be considered. However, itonly binds these ions at strong alkaline pH (therefore it is now rarelyused as intracellular Ca indicator). A more potent intracellular calciumchelator BAPTA/AM in fact has a neuroprotective effect by preventing thecell damage caused by elevated cytoplasmic Ca levels (Collatz et al.1997) during cerebral ischemia. Moreover, the cytotoxicity of Calcein AMin cancer cells that we observed depends on TopBP1, and Calcein AMtreatment induces E2F1 and mutp53 target gene changes. Thus, it isunlikely that the cytotoxicity is caused by calcium chelation. Anotherpotential property of Calcein AM warrants consideration is itssensitivity to ROS (reactive oxygen species). After removal of theacetomethoxy side chain, calcein can be oxidized and becomesfluorescent. Thus it was proposed to be used as a detector ofintracellular oxidative activity (Uggeri et al. 2004). However, theROS-sensitive fluorescein derivatives do not produce oxidative damage(instead they recycle ROS), therefore are used to detect intracellularROS generation in living cells (Molecular Probes™). Lack of pS15-p53signal after Calcein AM treatment (see Supplementary FIG. 9 in(Chowdhury et al. 2014)) also indicates that it does not produceoxidative damage or DNA damage.

TopBP1 protein is overexpressed in nearly 60% (46 out of 79 cases) ofprimary breast cancer tissues when compared with matched uninvolvedbreast tissues by Western and IHC analyses (Liu et al. 2009). Anincreased risk of breast cancer relapse or death is seen in patientswith tumors containing high levels of TopBP1 protein (Liu et al. 2009)or TopBP1 RNA (analysis of four breast databases composed of 1028patients (Liu et al. 2011)). Activation of PI(3)K/Akt signaling occursin 77% of breast cancer (see reference in (2012)). Thus, this convergentevent is expected to be quite prevalent in breast cancer, particularlyin TNBCs which overexpress TopBP1 in nearly all cases (Liu et al. 2009)and harbor mutp53 in 84% of cases (see reference in (TCGA 2012)).Therefore, the impact of this novel therapy would be very significantfor many breast cancer patients. Since these oncogenic pathways arecommon to many cancers. For example, the genetic alterations inglioblastoma occur mainly in these three signaling pathways:RTK/RAS/PI(3)K (88%), p53 (87%) and Rb (78%) (see reference in (TCGA2008)). Mutations in p53 and PI3K/Akt pathways are among the mostfrequent genetic abnormalities in all cancers and are stronglyassociated with poor prognosis, and resistance to therapy. Hence, theanti-TopBP1 therapy might be applicable to many other types of cancers.

Accordingly, the present invention provides a method of treating cancerthat overexpresses TopBP1 by administering to a patient suffering fromthe cancer with an effective amount of a small molecule inhibitor thatbinds the BRCT7/8 domain of TopBP1.

The small molecule inhibitor may be Calcein or Calcein AM. The cancerthat overexpresses TopBP1 may be identified by immunohistochemistry,immunoblotting or measurement of TopBP1 mRNA levels by quantitativeRT-PCR (reverse transcription-polymerase chain reaction). See, forexample, K. Liu, et al. (Liu et al. 2009), “Regulation of p53 by TopBP1:a Potential Mechanism for p53 Inactivation in Cancer,” Molecular andCellular Biology, May 2009, pages 2673-2693. The effective amount of thesmall molecule inhibitor that binds the BRCT7/8 domain of TopBP1 andblocks its functions can be determined by pS1159 peptide binding andGST-p53(DBD) pulldown assays. In the case where Calcein is used, itappears that the effective amount of Calcein that needs to be present is60-300 nM. See FIG. 1, f and g.

In one embodiment of the inventive method of treating cancer thatoverexpresses TopBP1, the cancer to be treated is selected from thegroup consisting of breast cancer, ovarian cancer, leukemia, lungcancer, multiple myeloma, hepatoma and gliobastoma multiforme.

In another embodiment, the method of treating cancer that overexpressesTopBP1 further comprises the step of administering to the patient achemotherapeutic agent.

In another embodiment, the invention provides a method of sensitizingcancer cells to a chemotherapeutic agent in a patient suffering fromcancer by administering an effective amount of a small moleculeinhibitor that bind the BRCT7/8 domain of TopBP1. The small moleculeinhibitor may be selected from the group consisting of Calcein andCalcein AM. The chemotherapeutic agent may be doxorubicin or cisplatin.

Other objects and features of the present invention will become apparentfrom the following detailed description considered in conjunction withthe accompanying drawings. It is to be understood, however, that thedrawings are designed solely for purposes of illustration and not as adefinition of the limits of the invention, for which reference should bemade to the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 Calcein binds TopBP1-BRCT7/8 and inhibits its binding to thepS1159 peptide and p53-DBD.

-   -   (a) Structure of calcein, a fluorescent molecule with formula        C₃₀H₂₆N₂O₁₃ and molecular weight 622.55.    -   (b) Purified TopBP1-BRCT7/8 was incubated with DMSO or calcein        and Btn-pS1159 peptide (pP) or nonphosphorylated peptide (nP),        followed by streptavidin Sepharose pulldown as described in the        Methods (Liu et al. 2006). The pulldown TopBP1-BRCT7/8 was        analyzed by Western blotting (IB) with a TopBP1 antibody (BL893,        Bethyl Laboratories) which recognizes the C-terminus of TopBP1.        The result was replicated three times.    -   (c) Purified TopBP1-BRCT7/8 was incubated GST-p53(DBD) in the        presence of DMSO or calcein, and GST pulldown assay was        performed (Liu et al. 2009). This result was replicated four        times.    -   (d) The lowest-energy docking position of calcein (ball & stick        model) in the structure of TopBP1-BRCT7/8 (shown as an        electrostatic molecular surface), viewed from two different        angles, shows favorable multiple polar and hydrophobic        interactions between BRCT7/8 and calcein with a large aromatic        system.    -   (e) The interactions between the docked structure of calcein and        the adjacent residues of TopBP1. H-bonds are shown as dotted        lines.    -   (f) Streptavidin Sepharose pulldown as in (b) was performed in        the presence of various concentrations of calcein.    -   (g) GST-p53(DBD) pulldown assay as in (c) was performed in the        presence of various concentrations of calcein.

FIG. 2 Calcein inhibits TopBP1 oligomerization and binding to E2F1 andmutp53 and disrupts the interaction of mutp53 from p73.

-   -   (a) MDA-MB468 cells were transfected with Myc-TopBP1 alone and        in combination with FLAG-TopBP1-BRCT7/8. Cells were treated with        Calcein AM (2.5 μM) or DMSO for 5 h. Cells were then lysed and        immunoprecipitated with anti-FLAG beads followed by        immunoblotting as described (Liu et al. 2013).    -   (b) MDA-MB468 cells were treated with Calcein AM (2.5 μM) or        DMSO for 5 h, and then lysed and incubated with 20 mM DMP for 30        min following the cross-linking protocol described in the        Methods section. Cell lysates were analyzed by SDS-PAGE and        immunoblotted with indicated antibodies. MK-2206 (1 μM for 24 h)        served as a positive control (Liu et al. 2013). The result was        replicated for two times.    -   (c) HEK293 cells expressing Myc-TopBP1 and FLAG-E2F1 were        treated with Calcein AM or DMSO for 5 h, followed by        immunoprecipitation and immunoblotting as indicated.    -   (d) MDA-MB468 cells were treated with Calcein AM (2.5 μM) or        DMSO for 5 h. The cell lysates were then subjected to        immunoprecipitation with anti-TopBP1 antibody or control mouse        IgG and then immunoblotting to detect interacting mutp53 and        E2F1. This result was replicated for three times.    -   (e) MDA-MB468 cells were treated with Calcein AM (2.5 μM) or        DMSO for 5 h. The cell lysates were then subjected to        immunoprecipitation with anti-p73 antibody or control mouse IgG        and then immunoblotting to detect mutp53.    -   (f) BT549 cells were transfected with FLAG-TopBP1 or an empty        vector. Two days later, cells were treated with Calcein AM (2.5        μM) or DMSO for 3 h and then harvested for anti-FLAG        immunoprecipitation (Liu et al. 2013).    -   (g) The interaction of endogenous TopBP1 and mutp53 in BT549        cells was examined by co-immunoprecipitation using anti-TopBP1        antibody or a control mouse IgG after cells were treated with        Calcein AM (2.5 μM) or DMSO for 5 h.    -   (h) BT549 cells were treated with Calcein AM (2.5 μM) or DMSO        for 5 h, and then subjected to anti-p73 (or a control mouse IgG)        immunoprecipitation.

FIG. 3 Calcein AM induces E2F1 and p53 activities and blocks mutp53 gainof function.

-   -   (a) E2F1 transcriptional activity was measured by a p14^(Arf)        promoter dual luciferase assay in HEK293 cells (Liu et al. 2003)        after Calcein AM treatment. The normalized activities of E2F1        were shown as fold induction relative to that of the empty        vector control. Results shown are the means±standard deviation        (S.D.) from three independent experiments (biological        replicates). *, P<0.001 (two-tailed t test).    -   (b) p53 transcriptional activity was measured by a p21 promoter        dual luciferase assay in H1299 cells (Liu et al. 2009) after        Calcein AM treatment. The normalized activities of p53        (means±S.D. from three biological replicates) were shown as fold        induction relative to that of the empty vector control. *,        P<0.05 (two-tailed t test).    -   (c) The effect of Calcein AM on mutp53(R273H) gain of function        was measured in p53-null H1299 cells expressing p73α and mutp53        (R273H), along with a p73 activity reporter plasmid (a Bax        promoter-luciferase plasmid) and pRL-TK as described (Liu et al.        2011). Luciferase activity of transfected p73 was determined as        fold induction relative to that of the empty vector control        (means±S.D. from three biological replicates). A portion of the        cell lysates was subjected to immunoblotting with the indicated        antibody (upper panel). *, P<0.001 (two-tailed t test).    -   (d) MDA-MB468 cells were treated with Calcein AM as in FIG. 2d .        RNA was then isolated for real-time RT-PCR analysis using        primers specific to E2F1, p53 and NF-Y target genes or GAPDH.        Results were normalized to GAPDH levels and the means±S.D. (n=3)        are expressed relative to the expression of genes in control        cells. Aliquots of the cell lysates were analyzed by        immunoblotting (right panel). The experiment was performed in        biological triplicates. **, P<0.001; *, P<0.01; #, P<0.05        (two-tailed t test).

FIG. 4 Calcein AM shows anti-tumor activity against many differentcancer cell lines.

-   -   (a) MTS assays were performed in breast cancer cell lines        MDA-MB468, MDA-MB231 and MCF7, and non-transformed breast        epithelial cell line MCF10A with increasing concentrations of        Calcein AM for 5 h. The data represent means±S.D. (n=3        biological replicates). The immunoblot showed the TopBP1        expression levels in these four cell lines (upper panel).    -   (b) Cells were treated with Calcein AM (2.5 μM) overnight and        then subjected to Caspase-Glo 3/7 activity assay. Shown are        means±S.D. derived from three independent experiments. *,        P<0.001, compared with DMSO control (two-tailed t test).    -   (c) The effect of Calcein AM (2.5 μM) on the doxorubicin        sensitivity in MCF10A and MDA-MB468 cells lines. MTS assays were        performed 5 h after treatment. The 490 nm absorbance readings        were normalized to the “0 μM Doxorubicin” controls within each        group. The MTS readings normalized to the “DMSO, 0        μM-Doxorubicin” control cells are presented in Supplementary        FIG. 5 in paper (Chowdhury et al. 2014). Shown are means±S.D.        derived from three biological replicates. P<0.001 compared with        DMSO control.    -   (d) MDA-MB468 cells were seeded into an Ultra-low Adherent        6-well plate with complete MammoCult™ medium, and then cultured        for 11 days in DMSO or Calcein AM (2.5 μM). The mammospheres        were observed at 10× magnification. The diameters of 15        mammospheres were measured from three independent culture        experiments using a Zeiss digital inverted microscope (Axio        Observer). *, P<0.001 (two-tailed t test). Higher-magnification        images are shown in Supplementary FIG. 6 (Chowdhury et al.        2014).    -   (e) MDA-MB468 cells were treated with DMSO or increasing doses        of Calcein AM for 16 h. Cells were then grown in fresh media        without Calcein AM for eight more days and then stained with        crystal violet. Each treatment was performed in triplicate.        Representative images are shown in the left panels (a complete        set of data are shown in Supplementary FIG. 7 (Chowdhury et al.        2014)). Colonies were counted and colony numbers relative to        DMSO controls (means±S.D.) are shown in the right graph. *,        P<0.001 compared with DMSO controls (two-tailed t test).    -   (f) In parallel with the experiment in (e), some MDA-MB468 cells        were treated with DMSO or increasing doses of Calcein AM for 16        h, and then assayed for caspase activation. Shown are means±S.D.        derived from three biological replicates. *, P<0.001, compared        with DMSO control (two-tailed t test).

FIG. 5 Calcein AM sensitizes mutp53-harboring cancer cells tochemotherapy.

-   -   (a) Cell viability was measured by MTS assay in an ovarian        cancer cell line SKOV-3 with increasing concentrations of        Calcein AM for overnight.    -   (b) SKOV-3 cells were treated with cisplatin (20 μM) alone or        with Calcein AM (5 μM) for 20 h, and then subjected to MTS        assay.    -   (c) SKOV-3 cells (p53-null) were transfected with an empty        vector control or mutp53(R273H) and then treated with cisplatin        (20 μM) alone or with Calcein AM (5 μM) for 20 h. Apoptosis was        measured by Caspase-Glo 3/7 activity assay. Expression of mutp53        was confirmed by Western blot analysis (right panel).    -   (d) RPMI 8226 cells were treated with an increasing dose of        doxorubicin along with vehicle (DMSO) or with Calcein AM (2.5        μM) for 12 h before subjecting to MTS assay. Each treatment was        tested in triplicate, and the values were normalized to vehicle        control wells. Results shown in (a), (b), (c) and (d) are        means±S.D. derived from three biological replicates. P values        are for two-tailed t test.

FIG. 6 Calcein AM sensitivity correlates with TopBP1 expression and Aktactivity in NCI-60 cancer cell lines.

-   -   (a) The sensitivity to Calcein AM (NSC 689290) and TopBP1 mRNA        levels (GC181500) and pT308-AKT levels by RPPA (MT18349) in        NCI-60 cell lines were extracted from NCI-DTP server. Box lines        highlight the cell lines with discrepancy in Log GI₅₀ and TopBP1        mRNA levels. Most of the discrepancy was found in cells with low        pT308-AKT levels except HOP-92 cells (dotted box).    -   (b) The correlation between the sensitivity of NCI-60 cell lines        to Calcein AM (expressed as Log GI₅₀, the concentration that        causes 50% growth inhibition) and TopBP1 RNA levels in these        cell lines. Pearson correlation coefficient (r) and P value are        shown.    -   (c) Only the top 30 cell lines with a higher level of pT308-AKT        are included in this correlative analysis.    -   (d) The NCI-60 cell lines were ranked according to either        pT308-AKT or total AKT expression levels followed by the        correlative analysis with TopBP1 expression for the selected        cell lines.

FIG. 7. The cytotoxicity of Calcein AM in cancer cells depends on E2F1and p63/p73.

-   -   (a) BT549 cells were treated with Calcein AM at indicated        concentrations for 16 h. Apoptosis was then analyzed by        Caspase-Glo 3/7 activity assay. Shown are means±S.D. derived        from three biological replicates. **, P<0.01; ***, P<0.001,        compared with DMSO control (two-tailed t test).    -   (b) BT549 cells were transfected with two shRNAs against E2F1 or        a scrambled (Scr) shRNA. 48 h after transfection, cells were        treated with Calcein AM (2.5 μM) for 16 h and then subjected to        Caspase-Glo 3/7 activity assay. Shown are means±S.D. derived        from three biological replicates. #, P<0.001 compared with DMSO        group; *, P<0.05; **, P<0.01, compared with shScr-Calcein AM        group (two-tailed t test). Depletion of E2F1 in these samples        was confirmed by Western blot analysis (right panels).    -   (c) BT549 cells were transfected with two shE2F1 as in (b), and        then treated with Calcein AM (2.5 μM) or DMSO for 20 h. Total        number of living cells was counted by trypan blue exclusion        assay and normalized to the vehicle controls. Shown are        means±S.D. derived from three biological replicates. **, P<0.005        compared with shScr-Calcein AM group (two-tailed t test).    -   (d) BT549 cells were transfected with shRNA against p63 or a        scrambled (Scr) shRNA. 48 h after transfection, cells were        treated with Calcein AM (2.5 μM) for 17 h and then subjected to        Caspase-Glo 3/7 activity assay. Shown are means±S.D. derived        from five biological replicates. #, P<0.001 compared with DMSO        group; ***, P<0.001 compared with shScr-Calcein AM group        (two-tailed t test). Depletion of p63 was confirmed by Western        blot analysis (upper panels).    -   (e) The expression of p73 in BT549 was depleted by transfecting        shRNA against p73. Two days later, cells were treated with        Calcein AM (2.5 μM) for 17 h and then subjected to Caspase-Glo        3/7 activity assay. Shown are means±S.D. derived from three        biological replicates. #, P<0.001 compared with DMSO group; **,        P<0.01 compared with shScr-Calcein AM group (two-tailed t test).        Depletion of p73 was confirmed by Western blot analysis (upper        panels).    -   (f) BT549 cells were transfected with shp63 or shp73 as in (d)        and (e), and then treated with Calcein AM (2 μM) or DMSO vehicle        for 15 h. The number of viable cells was counted by trypan blue        exclusion assay and normalized to the vehicle controls. Shown        are means±S.D. derived from three biological replicates. The        result was replicated two times. ***, P<0.001 compared with        shScr-Calcein AM group (two-tailed t test).

FIG. 8 The sensitivity to Calcein AM in cancer cells depends on TopBP1.

-   -   (a) MDA-MB231 cells were infected with recombinant adenoviruses        expressing empty vector (AdCMV) or AdTopBP1 (multiplicity of        infection 500). After two days, the sensitivity to doxorubicin        in the presence of DMSO or Calcein AM (2.5 μM) was measured as        in FIG. 4c . Shown are means±S.D. from three biological        replicates. TopBP1 overexpression was confirmed by Western        blotting (right panel). The 490 nm absorbance readings were        normalized to the “0 μM Doxorubicin” controls within each group.        The MTS readings normalized to the “DMSO, 0 μM-Doxorubicin”        control cells are presented in Supplementary FIG. 8a (Chowdhury        et al. 2014).    -   (b) Doxorubicin sensitivity was assayed in TopBP1-depleted        MDA-MB468 cells treated with doxorubicin and either DMSO or        Calcein AM (2.5 μM) for 5 h and then subjected to MTS assay.        Shown are means±S.D. from three biological replicates. TopBP1        knockdown was confirmed by Western blotting (right panel). n.s.:        not significant. The MTS readings are relative to the “0 μM        Doxorubicin” controls of each group. The MTS readings normalized        to the “DMSO, 0 μM-Doxorubicin” control cells are presented in        (Chowdhury et al. 2014) Supplementary FIG. 8b.    -   (c) HEK293 cells transfected with pSuper-shScr or two different        pSuper-shTopBP1 constructs (shTop #1 and #2) (Liu et al. 2004)        were treated with 2.5 μM Calcein or DMSO overnight. Apoptosis        was measured by Caspase-Glo 3/7 activity assay. *, P=0.001,        compared with shScr control group (two-tailed t test, n=3        biological replicates). E2F1 expression and depletion of TopBP1        were shown by immunoblotting (right panel).

FIG. 9 Effect of Calcein AM on tumor growth in the MDA-MB468 xenograftmodel and impact of TopBP1 on Calcein AM response in cultured MDA-MB468cells.

-   -   (a) Nude mice bearing MDA-MB468 xenografts were administered        with Calcein AM or vehicle DMSO via i.p. injection. Mean tumor        volumes±S.D. are shown; n=5 mice per group. This result was        replicated, and shown in (Chowdhury et al. 2014) Supplementary        FIG. 11.    -   (b) Photographs of the MDA-MB468 xenograft tumors (left), and        tumor weights (right).). *, P<0.001 (two-tailed t test).    -   (c) Mean mouse body weights±S.D. (n=5) from the injection of        MDA-MB468 cells (day −11).    -   (d) Representative images of H&E, Ki-67 immunohistochemical        staining and TUNEL staining of xenografts at 10× magnification        (upper panels). Representative images of H&E, Ki-67, TUNEL, and        cleaved caspase-3 staining at 40× magnification (lower panels).    -   (e) The effect of Calcein AM in MDA-MB468 cells depended on        TopBP1. TopBP1-depleted MDA-MB468 cells were treated with        Calcein AM (2.5 μM) overnight and then subjected to Caspase-Glo        3/7 activity assay. siScr: scrambled RNAi control. *, P=0.002,        compared with siScr/DMSO, P=0.01 compared with siTopBP1/Calcein        AM (two-tailed t test, n=3). Depletion of TopBP1 was verified by        Western blot analysis (upper panel).    -   (f) TopBP1-depleted and the siScr control MDA-MB468 cells were        treated with Calcein AM (2.5 μM, 5 h), and E2F1, p53 and NF-Y        target gene expression was analyzed by qRT-PCR as in FIG. 3d .        **, P<0.01, *, P<0.05, compared with DMSO samples; ##, P<0.01,        #, P<0.05, compared with siScr/Calcein AM samples (two-tailed t        test). The data represent means±S.D. (n=3).    -   (g) Aliquots of the cell lysates in (f) were analyzed for        Western blotting.

FIG. 10 Role of E2F1 in mediating growth suppression upon TopBP1depletion and a scheme summarizing the present studies.

-   -   (a) The decrease in cell number upon TopBP1 depletion is in part        dependent on E2F1. We first established U2OS stable cell lines        for doxycycline-inducible expression of control shGFP, shTopBP1,        and both shTopBP1 and shE2F1 using T-REX system (Yang et al.        2008; Liu et al. 2009; Wang et al. 2010). ShRNAs were then        induced with doxycycline (DOX) on day 0 (pink lines), and cells        were counted by a Beckman Coulter counter. An aliquot of        DOX-treated shTopBP1 cells were plated in medium without        doxycycline (Dox withdrawal, W/D) since day 13 (third graph from        the left, Dox withdrawal, green line). To make it easy to        compare, day 0-17 of shTopBP1 was plotted (second graph from the        left) for comparison with shGFP, and day 0-27 of shTopBP1 was        plotted (third graph from the left) for comparison with        shTopBP1/shE2F1. The data represent means±S.D. from three        biological replicates. *, P<0.001 when compared with “no Dox”,        and P<0.01 when compared with “Dox withdrawal” (two-tailed t        test). Lower panels: Western blots of TopBP1 and E2F1.    -   (b) TopBP1 is at a convergent point between Rb and PI(3)K        signaling pathways and contributes to tumor progression through        E2F1 and p53 regulation. Its action is blocked by Calcein AM.

FIG. 11 High TopBP1 expression in acute myelogenous leukemia (AML)patients is associated with a poor prognosis.

The expression of TopBP1 in AML samples and patients' overall survivalfrom a database (Verhaak et al. 2009) are analyzed. All patients werefirst ranked according to their TopBP1 expression in leukemia samples,and then grouped to three groups: 1. low TopBP1 expression group (thebottom 0-15% of the patients), 2. intermediate TopBP1 expression group(15-85%), and 3. high TopBP1 expression group (the top 85-100% of thepatients).

FIG. 12 High TopBP1 expression in cytogenetically normal-acutemyelogenous leukemia (CN-AML) patients is associated with a shortersurvival.

The expression of TopBP1 in CN-AML samples and patients' overallsurvival from a database (Metzeler et al. 2008) are analyzed. Allpatients were first ranked according to their TopBP1 expression inleukemia samples, and then grouped to two groups: 1. low TopBP1expression group (the bottom 0-49% of the patients), and 2. high TopBP1expression group (the top 49-100% of the patients).

FIG. 13 Calcein AM is active in killing an established T cell leukemiacell line (Jurkat-T cell) and primary leukemia cells prepared from apatient with T cell acute lymphoblastic leukemia.

Jurkat-T cells and primary leukemia cells prepared from a patient with Tcell acute lymphoblastic leukemia were treated with Calcein AM at 2.5 μMfor 7 hours and then the induction of cell apoptosis was analyzed bycaspase 3/7 activation assay. *, p<0.005, (compared with DMSO group)

FIG. 14 Calcein AM induced apoptosis in glioblastoma multiforme (GBM)cells (U-87 MG) and lung cancer cells (NCI-H520).

U-87 cells (GBM cells) or NCI-H520 cells (lung cancer cells) weretreated with Calcein AM at 2.5 or 5 μM for 16 hours and then theinduction of cell apoptosis was analyzed by caspase 3/7 activationassay. *, p<0.005, (vs. DMSO group).

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Methods

Cell Culture and Transfection

Human embryonic kidney cell lines HEK293 and HEK293T, human breastcancer cell lines MDA-MB468, MDA-MB231 and MCF7, human ovarian cancercell line SKOV-3, human non-small cell lung cancer cell line NCI-H1299,mouse mammary carcinoma cell line 4T1 and rat hepatoma cell lineMcA-RH7777 were maintained in DMEM with 10% FBS. MCF10A, a normal humanbreast epithelial cell line, was maintained in DMEM/F12 with 5% horseserum, 2.5 mM glutamine, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 100ng/ml cholera toxin and 20 ng/ml EGF. Human breast cancer cell lineBT549 and human myeloma cell line RPMI 8226 cells were maintained inRPMI with 10% FBS. All cell lines were obtained from ATCC and weretested negative for mycoplasma contamination. For maintenance, all celllines were kept in 37° C. humidified incubator with 5% CO₂. For HEK293,HEK293T and NCI-H1299 cells standard calcium phosphate method was usedfor transfection. Polyethylenimine (PEI) was used to transfect MDA-MB468cells and in some experiments of HEK293T cells. SKOV-3 cells weretransfected by electroporation (Bio-Rad). BT549 cells were transfectedusing the Gene Pulser Xcell Electroporation System or PEI.

Virtual Screening and Docking

Schrödinger suite (version 2010, Schrödinger, LLC, New York, N.Y.,2010), which includes all of the programs described below, was used tocarry out molecular modeling and docking studies. TopBP1-BRCT7/8 protein(PDB code: 3AL3) was prepared using the protein preparation wizard inMaestro 9.1 with default protein parameters: water molecules wereremoved, hydrogen atoms added and the BACH1 peptide ligand extracted fordocking H-bonds were optimized and the protein was energeticallyminimized using OPLS2005 force field. A receptor grid, which is largeenough to contain the whole active site, was generated using the programGlide without constraints.

2,000 compounds from Microsource Spectrum Collection were used for thevirtual screening. Compound structures were prepared using the programLigPrep and then docked into the protein using Glide (dockingparameters: standard-precision and dock flexibly). The top 100 compoundswere analyzed manually, among which 61 compounds with M.W. greater than250 were purchased from commercial vendors for further testing.

In Vitro Peptide Binding

The GST fusion proteins in Escherichia coli strain BL21 were induced by0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and purified accordingto the standard protocol. The GST portion on GST-TopBP1-BRCT7/8 wasexcised by Pre-Scission protease (Pharmacia). Two purified (>98%) biotinconjugated peptides were synthesized from Sigma Genosys. The peptidesequence is derived from the surrounding sequence of TopBP1 S1159: pP,Btn-REERARLApSer¹¹⁵⁹NLQWPS and nP, Btn-REERARLASNLQWPS. PurifiedTopBP1-BRCT7/8 (2 μg) was incubated with compounds in NETN-A buffer (50mM NaCl, 1 mM EDTA, 20 mM Tris, 0.5% NP-40) at 4° C. for 3 h withconstant rotation. Then 2 μg of purified pP or nP peptides were addedand rotated for another 3 h at 4° C. The biotin-conjugated peptides werethen pulled down with streptavidin-Sepharose (Amersham). The beads werewashed six times with NETN-B buffer (100 mM NaCl, 1 mM EDTA, 0.2 mMphenylmethylsulfonyl fluoride) and then subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed byWestern blotting with anti-TopBP1 antibody (1:1000, BL893, BethylLaboratories).

GST Pull-Down Assay

Purified TopBP1-BRCT7/8 (2 μg) was incubated with compounds in NETN-Abuffer as above at 4° C. for 3 h with constant rotation. Then purifiedGST-p53-DBD or GST (2 μg) were added and rotated for another 3 h at 4°C. GST-p53-DBD was then pulled down with glutathione-Sepharose. Thebeads were washed six times with NETN-B buffer and then subjected toSDS-PAGE analysis.

Immunoprecipitation and Western Blot Analysis

MDA-MB468 cells were lysed with TNN buffer (Liu et al. 2003). An aliquotof cell lysates was lysed with SDS lysis buffer and the rest of celllysates were incubated with appropriate antibodies or beads at 4° C. for3-12 h. Anti-FLAG beads (Sigma) were washed three times with TNN buffer.Immunoprecipitates were fractionated by SDS-PAGE and electro-transferredto Immobilon-P membrane (Millipore). Equal amount of protein loading wasvalidated with Ponceau-S staining. The specific signals were detected byincubating with appropriate antibody. All primary antibodies were usedat 1:1000 dilution and horseradish peroxidase (HRP)-conjugated secondaryantibodies were used at 1:5000 dilution for immunoblotting. E2F1 (C-20and KH-95), p53 (FL393), p63 (4A4 and H-129), p73 (H-79), p21 (C-19),Bax (N-20), GST (B-14), c-Myc (A14), Miz1 (H-190), Chk1 (G4), Actin(C-2) and GAPDH (6C5) antibodies were purchased from Santa Cruz. TopBP1(mouse monoclonal) and Aid antibodies were purchased from BDTransduction Laboratories. TopBP1 (BL893, Rabbit polyclonal) antibodywas purchased from Bethyl. Mouse monoclonal anti-phospho-γH2AX waspurchased from Millipore. Phospho-Chk1 (Ser345) antibody was purchasedfrom Cell signaling. FLAG (F7425) antibody was purchased from Sigma.Mouse monoclonal anti-p73 (IMG-246) was purchased from IMGENEX.

Cross-Linking

Borate buffer (50 mM NaBorate, 100 mM potassium acetate, 2 mM MgCl₂, 1mM EGTA, 1% Trition X-100, and protease inhibitors [pH 8.57]) was usedto lyse MDA-MB468 cells on ice for 10 min. followed by 10 min high-speedcentrifugation to remove insoluble material, and then incubated with 20mM freshly prepared DMP (Dimethyl pimelimidate-2HCl, Thermo Scientific)for 30 min. An equal volume of 50 mM NH₄Cl in PBS was added andincubated for 10 min to stop the reaction. The cell lysates weresubjected to SDS-PAGE and Western blot analysis.

Luciferase Assay

E2F1 activity reporter assay using a p14^(ARF) promoter-luciferaseplasmid was performed as described (Liu et al. 2003). p53 activityreporter assay was performed by a p21^(Cip) promoter-luciferase plasmid(Liu et al. 2009). p73 activity reporter assay was performed using apGL3-Bax promoter-luciferase plasmid (Liu et al. 2011). pRL-TK (Renillaluciferase, Promega) was used in all experiments for controllingtransfection efficiency. The firefly and Renilla luciferase activitieswere measured by Dual-Luciferase Reporter System (Promega), and thefirefly luciferase activities were normalized against the Renillaactivity. All assays were performed in triplicate.

Real Time RT-PCR

RNA was isolated using TRIzol method (Invitrogen). Quantitative reversetranscription-PCR (RT-PCR) was performed in triplicate using the forwardand reverse primers ((Chowdhury et al. 2014) Supplementary FIG. 18) onan MX3005P thermal cycler using SYBR green dye method to observe theprogress of the reactions with ROX dye, which was added as reference.GAPDH was run in parallel with target genes.

MTS Assay

Cells (10⁴ cells/well) were plated into 48 well plates (BD Falcon) andtreated with DMSO or Calcein AM separately. Then 50 μl of MTS reagent(CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega)was added to each well, covered from light and incubated for 1 h at 37.0incubator to develop purple color. The samples were measured at 490 nmin a plate reader (BioTek Synergy HT) against a blank 48-well plate.Each assay was done in triplicate.

Caspase 3/7 Activation Assay

The apoptosis was measured by Caspase-Glo® 3/7 assay (Promega) followingthe manufacturer's protocol. The cells were plated in 6-well tissueculture dish (10⁴ cells/well) (BD Falcon) in respective media. Next daythe cells were treated with DMSO (as negative control) and Calcein AMfor 5 h. The blank reactions were prepared per direction. Equal volumeof Caspase-Glo® 3/7 reagents were applied to each well. A luminometer(Sirius) was used to quantitate luminescence.

Colony Formation Assay

MDA-MB468 cells were plated in 6-well plates at 200 cells/well. Thecells were either treated DMSO vehicle or treated with indicatedconcentration of Calcein AM for 16 h and then released by 3 times 1×PBSwashing. Cells were grown in fresh media without drugs for additional 8days to form colonies, when each colony contained more than 50 cells.Plates were then stained with 5% crystal violet (0.5 g Crystal violet,25 ml methanol and 75 ml H₂O). Cell number was counted.

Mammosphere Formation and Treatment

For in vitro mammosphere culture, ˜5000 MDA-MB468 cells/well were seededinto a Ultra-low Adherent 6-well plate (Stem Cell Technologies) with 2ml/well fresh complete MammoCult™ medium (Stem Cell Technologies)containing the MammoCult® Basal Medium, MammoCult® ProliferationSupplement, 4 μg/ml Heparin (Stem Cell Technologies) and fresh 0.48μg/ml Hydrocortisone. Cells were incubated in a 5% CO₂ incubator at 37°C. for 7-11 days. Both the untreated (DMSO treated) and CalceinAM-treated mammospheres were observed at 10× magnification and measuredon day 11 under a Zeiss digital Axio Observer inverted microscope. Eachtreatment was done in triplicate.

Gene Knockdown

To prepare TopBP1 knockdown stable cell line, MDA-MB468 cells wereinfected with lentivirus expressing TopBP1 shRNA (Liu et al. 2011) aswell as scramble shRNA, separately. The infected cells were grown for 48h followed by selection with puromycin (2 μg/ml). After selection, theknockdown was confirmed by Western blotting before further experiments.Two different pLKO-shTopBP1 (#1 and #2) constructs (Open Biosystems,RHS4533-NM_007027) were used to deplete TopBP1 in MDA-MB468 cells.Additional two pSuper-shTopBP1 constructs (Liu et al. 2004) were used toknock down TopBP1 in HEK293 cells. Two pSuper-shE2F1 constructs wereused to deplete E2F1: shE2F1#1 (described previously (Liu et al. 2004)),and shE2F1#2 (target sequence 5′-GACTGTGACTTTGGGGACC-3′). The targetsequence for pSuperior-shE2F1 is the same as pSuper-shE2F1#1. To depletep63, shp63alpha pLKO.1-puro (from Addgene, 19120) was used. TP73 MISSIONshRNA (shp73 pLKO.1-puro) was purchased from Sigma (TRCN00002722587).

Xenograft Experiment

Female nu/nu mice (4-5 weeks old) were purchased from Charles RiverLaboratories (Cambridge, Mass.). The animals were cared for andmaintained by the Animal Care and Use Committee of Baylor College ofMedicine. Freshly grown MDA-MB468 breast cancer cells [(8×10⁶) cells persite in 100 μl phosphate-buffered saline (PBS)] were injectedsubcutaneously into the right side of the flank of the 5-6 weeks oldmice. When tumors were measurable, the mice were randomly divided intotreatment and control groups. Calcein AM was dissolved in DMSO and wasgiven intraperitoneally at 40 mg/kg for 3 doses every 3^(rd) day. Thecontrol group mice were injected with vehicle (DMSO) in the same way.The mice were monitored thrice per week. The weight of the mice andtumor size were measured on the indicated day with a caliper andcalculated based on the formula π/6 (length×depth×width). The evaluatorwas blinded to the group allocation during monitoring. Animals weresacrificed in the indicated dates, and tumors were harvested, weighed,and further processed for histopathological analysis. All experimentswere performed under a Baylor College of Medicine Institutional AnimalCare and Use Committee (IACUC)-approved protocol and all experimentsconfirm to IACUC standards and ethical regulations.

Histology and Immunohistochemistry

Both control (DMSO) and Calcein AM treated tumor samples were placed incassettes and fixed in 10% neutral buffered formalin for overnight. Nextday the samples were sent to The Pathology and Histology Core of BaylorCollege of Medicine for further processing. The paraffin-embeddedsections were stained with H&E, Ki-67, TUNEL and anti-cleaved caspase-3using standard operating protocols.

Statistical Analyses

We performed two-tailed t test for comparisons of treatment groups. Pvalues less than 0.05 were considered statistically significant. Pearsoncorrelation coefficient was calculated to evaluate correlations betweenthe mRNA expression of TopBP1 and Log GI₅₀ of Calcein AM in NCI-60 celllines. The sample size of the xenograft study was chosen to detect 50%response rate of xenografts, and use 5% for type II error, then n=log0.05/log 0.5=4.3. Thus, using 5 animals per group will have greater than95% of statistical power.

Results

Calcein Blocks TopBP1 Oligomerization and Binding to E2F1 and p53.

Based on the mechanisms of action, we sought to design the TopBP1inhibitors capable of blocking TopBP1 oligomerization and itsinteraction with mutp53. Since the BRCT7/8 domains of TopBP1 areresponsible for both oligomerization and binding to p53 (Liu et al.2009), and the BRCT domains form structured binding pockets,TopBP1-BRCT7/8 would be the ideal domains for screening compounds thatblock TopBP1 binding to pS1159 peptide and mutant p53. Using theTopBP1-BRCT7/8 structure (Leung et al. 2011), we performed a moleculardocking screening to identify compounds that can bind to TopBP1-BRCT7/8.From a library of more than 2000 compounds including FDA-approved drugs(Microsource Spectrum Collection), we identified 61 compounds with topscores for fitting into the structural pocket of TopBP1-BRCT7/8 and withmolecular weights of greater than 250 g. We then performed biochemicalbinding assays (pS1159 peptide binding assay (Liu et al. 2013) andGST-p53(DBD) pulldown assay (Liu et al. 2009)) to test the ability ofthese 61 compounds to block the interactions between TopBP1-BRCT7/8 andpS1159 peptide or p53(DBD). The compounds that showed activities in theinitial screening were subjected to multiple independent binding assaysfor reproducibility. We identified an oxospiro-benzofuran derivativecompound, calcein (FIG. 1a ) that blocked the interactions ofTopBP1-BRCT7/8 with pS1159 peptide (FIG. 1b ) and p53(DBD) (FIG. 1c ).The docking of calcein into TopBP1-BRCT7/8 pocket viewed from twodifferent angles is shown in FIG. 1d &e. As seen from FIG. 1e , calceinis predicted to form H-bonds with K1317, R1280, S1273, S1274, R1314 andR1407, among which K1317 and S1273 had been demonstrated to be importantfor binding to pS1159 for oligomerization (Liu et al. 2013). We alsotested these candidates in E2F1 activity reporter assay (Liu et al.2003) and p53 activity reporter assay (Liu et al. 2009). Calcein is ahydrophilic fluorescent molecule that does not get into cells. Itsnon-fluorescent acetomethoxy derivative, Calcein AM (acetoxymethylester) can enter cells where it is hydrolyzed by esterases to calceinand retains inside the cells. We therefore used Calcein AM for cellculture experiments. Among these potential compounds, Calcein AM stoodout in its ability to induce both E2F1 ((Chowdhury et al. 2014)Supplementary FIG. 1) and p53 activities ((Chowdhury et al. 2014)Supplementary FIG. 2). Both pS1159 peptide binding and GST-p53(DBD)pulldown assays showed that calcein blocked the TopBP1/pS1159interaction (FIG. 1f ) and TopBP1/p53(DBD) interaction (FIG. 1g ) atsubmicromolar concentrations.

Ironically, Calcein AM is developed as a cell viability assay since onlylive cells can hydrolyze non-fluorescent Calcein AM into fluorescentcalcein and retain calcein inside the cells. Calcein AM treatment wasreported to have cytotoxic effect in some cancer cells (Liminga et al.1995; Jonsson et al. 1996; Liminga et al. 2000); nevertheless, itsdetailed mechanisms of action remain unclear. We used Calcein AM tofurther investigate the in vivo effect of calcein in TopBP1 signaling.Since the interaction between TopBP1-BRCT7/8 and pS1159 peptide mediatesTopBP1 oligomerization (Liu et al. 2013), Calcein AM was expected toblock TopBP1 oligomerization. Indeed, using differentially taggedTopBP1, we found that Calcein AM (2.5 μM) was able to inhibit TopBP1oligomerization in triple-negative, PTEN-null MDA-MB468 breast cancercells (FIG. 2a ). Calcein AM also blocked endogenous TopBP1oligomerization in a DMP chemical crosslinking assay (Liu et al. 2013)(FIG. 2b ). Here MK2206, an Akt allosteric inhibitor was used as apositive control for blocking Akt-dependent TopBP1 oligomerization (Liuet al. 2013). Importantly, Calcein AM not only inhibited the interactionbetween overexpressed E2F1 and TopBP1 in HEK293T cells (FIG. 2c ), butalso inhibited TopBP1/E2F1 and TopBP1/mutp53 interactions in MDA-MB468cells (harboring mutp53(R273H)) at physiological levels (FIG. 2d ).TopBP1/mutp53 interaction has been shown to facilitate the binding ofmutp53 to p63/p73 (Liu et al. 2011). Calcein AM treatment also led torelease of p73 from mutp53 (FIG. 2e ).

Previously, we showed that TopBP1 can interact with both “contactmutant” p53 (such as R273H) and conformational mutants (such as R175Hand R249S) (Liu et al. 2011). Thus, we also tested Calcein AM in anothertriple-negative, PTEN-null breast cancer cell line BT549, which containsa conformational mutant R249S mutp53. Calcein AM treatment inhibited theinteraction between TopBP1 and mutp53 and E2F1 in BT549 cells (FIG. 2f&g). Consistent with a role for TopBP1 oligomerization (Liu et al.2006), Calcein AM also inhibited the interaction between TopBP1 and Miz1(FIG. 2f ), suggesting that the release of Miz1 from TopBP1 mightmediate some of Calcein AM activities. Importantly, Calcein AM treatmentalso resulted in release of p73 from mutp53(R249S) in BT549 cells (FIG.2h ) like it did in MDA-MB468 cells.

Calcein AM Activates E2F1 and p53 and Blocks Mutp53 GOF.

Next, we examined the effect of Calcein AM on E2F1 and p53transcriptional activities using E2F1 (Liu et al. 2003) and p53 activityreporter assay (Liu et al. 2009), respectively. In line with its effecton blocking TopBP1 function in repressing E2F1 and p53 activities,Calcein AM treatment enhanced E2F1 and p53 transcriptional activities(FIG. 3a-b ). To demonstrate the induction of p14^(Arf)promoter-luciferase activity by Calcein AM was indeed due to E2F1, weperformed an independent assay using a pair of the p68 subunit of DNApolymerase a promoter constructs: pKL12 (containing two wild-type E2Fsites) and pKL12-E2FAB (both E2F sites being mutated and no longerresponding to E2F) (Nishikawa et al. 2000; Liu et al. 2003). Indeed,Calcein AM could only induce the activity on pKL12, but not onpKL12-E2FAB ((Chowdhury et al. 2014) Supplementary FIG. 3). To show theinduction of p21 promoter activity by Calcein AM was dependent on p53,we used a pair of isogenic cell lines (Bunz et al. 1999) p53^(+/+) andp53^(−/−) HCT116 and showed that the induction of p21 and another p53target Bax was observed on in p53^(+/+), but not in p53^(−/−) HCT116cells ((Chowdhury et al. 2014) Supplementary FIG. 4). Previously using ap73 activity reporter (Bax-promoter-driven luciferase) assay, we foundthat in p53-null H1299 cells, overexpression of mutp53(R273H) blockedthe p73 transcriptional activity in a TopBP1-dependent manner (Liu etal. 2011). Consistently, mutp53(R273H) inhibited p73 transcriptionalactivity, and indeed this activity was blocked by Calcein AM (FIG. 3c ),indicating an inhibitory effect of Calcein AM on mutp53 GOF. To furtherinvestigate whether Calcein AM affected endogenous activities of E2F1and mutant p53, we examined the target genes of E2F1, p63/p73 and NF-Yin MDA-MB468 cells. Calcein AM up-regulated E2F1 pro-apoptotic targetgenes, such as p′73, Apaf1 and caspase 3 in MDA-MB468 cells (FIG. 3d ).Calcein AM also inhibited mutp53 GOF activities by up-regulation ofp63/p73 target genes such as Bax, Noxa, Hdm2 and down-regulation of NF-Ytarget genes, such as Cdk1, Cdc25C and Cyclin A2 (Liu et al. 2011) (FIG.3d ). These data demonstrate that calcein binds TopBP1 to block mutp53GOF and restore pro-apoptotic activities of E2F1 and p63/p73.

Calcein AM Kills TopBP1-Overexpressing Cancer Cells.

We then performed MTS assay to determine the cytotoxic activity ofCalcein AM in three breast cancer cell lines MDA-MB468, MDA-MB231, MCF7and a non-transformed MCF10A cell line. Calcein AM preferentiallyinhibited the proliferation of these three cancer cell lines (FIG. 4a ).The caspase 3/7 activity assay showed that Calcein AM induced apoptosisin MCF7, MDA-MB231 and MDA-MB468 cells, but to a much lesser degree inMCF10A cells (FIG. 4b ). When co-treated with doxorubicin (adriamycin),Calcein AM greatly enhanced the doxorubicin sensitivity in MDA-MB468,but not in MCF10A cells (FIG. 4c & (Chowdhury et al. 2014) SupplementaryFIG. 5). Mutp53 GOF has been shown to expand the breast cancer stemcells (Lu et al. 2013), which form mammospheres in suspension culture(Dontu et al. 2003). The mammosphere formation of MDA-MB468 cells wasalso blocked by Calcein AM (FIG. 4d ). It is possible that this is atleast in part due to the cytotoxic activity of Calcein AM, but at theconcentration used in this experiment, many cells still survived andcould be scored ((Chowdhury et al. 2014) Supplementary FIG. 6), but theyfailed to form mammosphere. To further investigate the cytostatic(inhibiting proliferation) and cytotoxic (inducing apoptosis) activitiesof Calcein AM in a quantitative way, we performed colony formation assay(FIG. 4e & (Chowdhury et al. 2014) Supplementary FIG. 7) and caspase 3/7activity assay (FIG. 4f ) after treating MDA-MB468 cells with increasingdoses of Calcein AM. While apoptosis was induced at 2.5 and 5 μM, butnot at 0.1 and 0.5 μM, inhibition of colony formation was apparent at0.1 μM and beyond. Thus, Calcein AM exerts its effect through itsactivity in suppressing proliferation and, when given at higher doses,inducing apoptosis.

We next extended the studies to other cancer cell lines. We found thatCalcein AM also inhibited the proliferation of p53-null SKOV-3 ovariancancer cells either alone (FIG. 5a ) or in combination with cisplatin(FIG. 5b ). Although overexpression of mutp53(R273H) inhibitedcisplatin-induced apoptosis in SKOV-3 cells (due to mutp53 GOF (Liu etal. 2011)), this effect could be partially rescued by Calcein AM (FIG.5c ). Calcein AM also sensitized cells to doxorubicin in a myeloma cellline RPMI 8226 harboring mutp53(E285K) (FIG. 5d ). Thus, Calcein AMappears to have activity against a broad spectrum of cancer cells. Infact, Calcein AM (NSC 689290) is among the active compounds in NCIDevelopmental Therapeutics Program (DTP) screening program, whichutilized a panel of NCI-60 cancer cell lines to test compounds at fiveconcentrations. The average Calcein AM GI₅₀ over all 60 cell lines is0.662 μM (ranging from 10 nM to 10.96 μM, see FIG. 6a ) (NCI-DTP data).The GI₅₀ of Calcein AM in several triple-negative breast cancer (TNBC)cell lines such as BT549 and Hs-578T, etc. are less than 0.4 μM, and inseveral lung cancer cell lines such as A549, NCI-H23, NCI-H460 andNCI-H522 are less than 1 μM (FIG. 6a ). We analyzed Calcein AMsensitivity (GI₅₀) and TopBP1 RNA levels in NCI-60 cell lines fromNCI-DTP server and found a correlation between high TopBP1 levels andthe sensitivity Calcein AM (r=−0.4439, two tailed p=0.0005, FIG. 6b ).Upon examining the Akt status (in RPPA database) of those cells withdiscordance (i.e. sensitivity does not correlate with TopBP1expression), we found most of them show low Akt activity (low T308phosphorylation), e.g. MDA-MB435 cells (DeGraffenried et al. 2004) (FIG.6a ). This suggested that the sensitivity to Calcein AM might beaffected by both TopBP1 levels and Akt activity. To test this in anunbiased manner, we ranked the NCI-60 cell lines according to eitherpT308Akt or total Akt levels, and calculated the Pearson correlationcoefficients. While total Akt status does not affect the correlationbetween TopBP1 and Calcein AM sensitivity, higher pT308Akt levels indeedshow better correlation (FIG. 6c-d ). Thus, Calcein AM has activityagainst a wide spectrum of cancer cells, and its sensitivity iscorrelated with TopBP1 levels, particularly in Akt-activated cancercells.

E2F1 and p63/p73 Mediate Calcein AM Anti-Cancer Activity.

Since Calcein AM activates E2F1 pro-apoptotic target genes, blocksmutp53 GOF (FIG. 3) and releases p73 from mutp53 binding (FIG. 2), weexamined whether the cytotoxic activity of Calcein AM was mediated byE2F1 and p63/p73 which were unleashed after Calcein AM interruptedTopBP1/E2F1 and TopBP1/mutp53 interactions. We chose BT549 for thesestudies due to their Calcein AM sensitivity and high transfectionefficiency. Consistent with the low GI₅₀ of Calcein AM (0.27 μM, fromNCI-DTP database, FIG. 6a ) for BT549 cells, Calcein AM inducedapoptosis in BT549 cells at 1 μM and above (FIG. 7a ). Depletion of E2F1by two shRNA constructs attenuated Calcein AM response as measured bycaspase activation (FIG. 7b ) and viability assay (FIG. 7c ). CalceinAM-induced apoptosis was also decreased by depletion of p63 (FIG. 7d )and p73 (FIG. 7e ). Correspondingly, there were more living cells inp63- and p73-depleted cells compared with scrambled shRNA after CalceinAM (FIG. 7f ). Together with E2F1 and p63/p73 target gene analysis shownin FIG. 3d , these data indicate that Calcein AM activates E2F1 andp63/p73 activities to induce apoptosis.

A Novel TopBP1-Dependent Mechanism for Calcein AM Activity.

With the correlation between TopBP1 levels and Calcein AM sensitivity inNCI-60 cancer cell lines (FIG. 6), we investigated how TopBP1 levelsaffected Calcein AM response. Overexpression of TopBP1 in MDA-MB231cells that express TopBP1 at lower levels rendered cells more resistantto doxorubicin. This result demonstrates the activity of TopBP1 inrepressing doxorubicin-induced apoptosis. Importantly, thisanti-apoptotic activity of TopBP1 was blocked by Calcein AM (FIG. 8a &(Chowdhury et al. 2014) Supplementary FIG. 8a). On the other hand,consistent with previous data on the up-regulation of E2F1- andp63/p73-dependent pro-apoptotic target gene expression by TopBP1knockdown (Liu et al. 2006; Liu et al. 2011), TopBP1 depletionsensitized MDA-MB468 to doxorubicin treatment (FIG. 8b & (Chowdhury etal. 2014) Supplementary FIG. 8b). While Calcein AM sensitized the shScrcontrol MDA-MB468 cells to doxorubicin, it failed to cast the sameeffect on TopBP1-depleted MDA-MB468 cells (FIG. 8b & (Chowdhury et al.2014) Supplementary FIG. 8b). Acute depletion of TopBP1 in HEK293 cellsinduced E2F1-dependent apoptosis (Liu et al. 2004). Calcein AM alsoinduced apoptosis in HEK293 cells (FIG. 8c ). In the TopBP1-depletedHEK293 cells where E2F1 activity has been unleashed, Calcein AM couldnot induce more apoptosis (FIG. 8c ). The data in FIG. 8a-c indicatethat Calcein AM blocks TopBP1 anti-apoptotic activity and that CalceinAM and TopBP1 shRNAs were targeting the same molecules or pathways.Together with the analysis of NCI-60 cells lines in FIG. 6, these datasupport TopBP1 as an in vivo target for Calcein AM. To rule out thepossibility that Calcein AM might cause DNA damage through otherundefined mechanisms, we treated MDA-MB468 with this compound up to 10μM and still did not see evidence of DNA damage as shown by lack ofeither pS15-p53 or γ-H2AX signal ((Chowdhury et al. 2014) SupplementaryFIG. 9). We also examined whether Calcein AM might interfere with TopBP1checkpoint activation. In fact, Calcein AM did not block HU-induced Chk1activation ((Chowdhury et al. 2014) Supplementary FIG. 10), and mightslightly promote Chk1 activation in a TopBP1-dependent manner. This isconsistent with the effect of Calcein AM in blocking TopBP1oligomerization and shifting TopBP1 toward monomeric form for checkpointactivation. Taken together, Calcein AM selectively kills cancer cellsthrough a TopBP1-dependent mechanism of action.

Calcein AM Inhibits the Growth of Breast Cancer Xenografts.

To investigate the in vivo activity of Calcein AM, we next establishedMDA-MB468 xenografts in nude mice and injected i.p. with Calcein AM (40mg/kg) or vehicle (DMSO) every three days for three doses (5 mice pergroup). Two independent experiments showed that Calcein AM significantlyinhibited xenograft growth (FIGS. 9a-b and (Chowdhury et al. 2014)Supplementary FIG. 11) as well as the expression of proliferation markerKi-67 (see Supplementary FIGS. 12-13 in paper (Chowdhury et al. 2014))without affecting body weights of mice (FIG. 9c and (Chowdhury et al.2014) Supplementary FIG. 11c). When the tumors started to regrow aroundday 60 in the experiment of (Chowdhury et al. 2014) Supplementary FIG.11, a second round of treatment was given at day 61 since. The tumorsresponded to Calcein AM injection again. Immunohistochemistry of thexenografts harvested one week after the second injection showedinhibition of proliferation and induction of apoptosis, as indicated byKi-67 staining, TUNEL assay and cleaved caspase 3 staining, respectively(FIG. 9d ).

We also tested the in vivo activity of Calcein AM against the xenograftformation in another cell line BT549. Twenty nude mice were injecteds.c. with BT549 cells. Four days later, mice were then injected i.p.either with DMSO (8 mice), Calcein AM 20 mg/kg (8 mice) or Calcein AM 40mg/kg (4 mice) every three days for three doses. Six out of eightDMSO-treated mice developed xenografts later. On the contrary, none ofthe eight mice injected with Calcein AM 20 mg/kg and none of the fourmice injected with Calcein AM 40 mg/kg developed tumors ((Chowdhury etal. 2014) Supplementary FIG. 14). Thus, Calcein AM has in vivo activityagainst tumor growth or development in MDA-MB468 and BT549 xenograftmodels.

The mice appeared to tolerate Calcein AM i.p. injections throughout thecourse of treatment. To investigate whether there was any acute toxicityin proliferating tissues, we injected Calcein AM 40 mg/kg and analyzedintestinal tissues 48 h after injection. As shown in (Chowdhury et al.2014) Supplementary FIG. 15, Calcein AM injection did not decrease Ki-67staining, nor did it induce apoptosis in the intestinal epithelium. Thelack of toxicity in normal mouse tissues could be in principle due to aspecies-specific activity of Calcein AM against human cells, but notmouse cells. While this is unlikely since TopBP1-BRCT7/8 is highlyconserved between human and mouse (74% identify, 83.3% similarity), andthe predicted calcein contact residues K1317, R1280, S1273, S1274, R1314and R1407 (FIG. 1e ) are 100% conserved in mouse TopBP1. To rule outthis possibility, we tested the activity of Calcein AM in mouse and rattumor cells. As shown in (Chowdhury et al. 2014) supplementary FIG. 16,Calcein AM also induced apoptosis in mouse breast tumor cells 4T1 andrat hepatoma cells McA-RH7777. Thus, Calcein AM selectively kills cancercells, but is not toxic to normal tissues.

We further utilized the MDA-MB468 cells expressing shTopBP1 to validatethe in vivo target of Calcein AM. Indeed, depletion of TopBP1 mitigatedthe effect of Calcein AM on apoptosis (FIG. 9e ; the results werereproduced in (Chowdhury et al. 2014) Supplementary FIG. 17 using twodifferent shTopBP1). Correspondingly, the induction of E2F1, p63/p73 andNF-Y target gene expression by Calcein AM treatment was less significantin these TopBP1-depleted cells (FIG. 9f ). We noted that in theTopBP1-depleted MDA-MB468 cells, E2F1 levels were reduced (FIG. 9g &(Chowdhury et al. 2014) Supplementary FIG. 17). It is possible thatacute depletion of TopBP1 induces E2F1-dependent apoptosis (Liu et al.2004), thus high E2F1 expressing cells would be counter-selected,resulting in selecting for the cells with lower E2F1 expression. Toinvestigate the role for E2F1 in mediating the effect of TopBP1depletion, we carried out doxycycline-inducible depletion of TopBP1 orboth TopBP1 and E2F1 in U2OS cells (FIG. 10a ) using U2OS T-REX system(Yang et al. 2008; Liu et al. 2009; Wang et al. 2010). The results showthat upon depletion of TopBP1 after adding doxycycline (to induce TopBP1shRNA), there was a decrease in cell number. This was a reversible andlikely regulatory effect by TopBP1, since the defect was immediatelyrescued by restoring TopBP1 expression upon withdrawal of doxycycline.Thus, it is unlikely due to structural genomic damage from TopBP1depletion. Importantly, the reduction in cell number by TopBP1 depletionwas largely rescued upon concurrent depletion of E2F1 (FIG. 10a ). Thisresult indicates that the role of TopBP1 in cell proliferation/apoptosisis in part mediated through its regulation on E2F1. Therefore, CalceinAM sensitivity is affected by TopBP1 and E2F1 levels in cancer cells.

Taken together, Calcein AM targets TopBP1/E2F1 and TopBP1/mutp53interactions and exerts its in vivo antitumor activity.

Thus, while there have shown and described and pointed out fundamentalnovel features of the invention as applied to a preferred embodimentthereof, it will be understood that various omissions and substitutionsand changes in the form and details of the devices illustrated, and intheir operation, may be made by those skilled in the art withoutdeparting from the spirit of the invention. For example, it is expresslyintended that all combinations of those elements and/or method stepswhich perform substantially the same function in substantially the sameway to achieve the same results are within the scope of the invention.Moreover, it should be recognized that structures and/or elements and/ormethod steps shown and/or described in connection with any disclosedform or embodiment of the invention may be incorporated in any otherdisclosed or described or suggested form or embodiment as a generalmatter of design choice. It is the intention, therefore, to be limitedonly as indicated by the scope of the claims appended hereto.

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We claim:
 1. A method of sensitizing cancer cells to a chemotherapeuticagent in a patient suffering from cancer by administering an effectiveamount of a small molecule inhibitor selected from the group consistingof Calcein and Calcein AM that binds the BRCT7/8 domain of TopBP1. 2.The method of claim 1 wherein the chemotherapeutic agent is doxorubicinor cisplatin.
 3. The method of claim 1, wherein the cancer cellsoverexpress TopBP1.